Enzyme Diversity of Nitrifying Organisms and Their Biotechnological Application
Diplomarbeit
zur Erlangung des akademischen Grades Master of Science in Engineering der Fachhochschule Campus Wien Master-Studiengang Bioverfahrenstechnik
Vorgelegt von: Mag. Dr. Andrea Kahlbacher, BSc Personenkennzeichen: c1810540006
FH-Hauptbetreuerin: Univ.-Prof. Dipl.-Ing. Dr. Kristina Djinovic-Carugo Zweitprüfer: Mag. Dr. Andreas Franz
Abgabetermin: 23.08.2020
FH Campus Wien
University of Applied Sciences/Fachbereich Bioengineering
FH Campus Wien
University of Applied Sciences/Fachbereich Bioengineering
Erklärung:
Ich erkläre, dass die vorliegende Diplomarbeit von mir selbst verfasst wurde und ich keine anderen als die angeführten Behelfe verwendet bzw. mich auch sonst keiner unerlaubten Hilfe bedient habe. Ich versichere, dass ich diese Diplomarbeit bisher weder im In- noch im Ausland (einer Beurteilerin/einem Beurteiler zur Begutachtung) in irgendeiner Form als Prüfungsarbeit vorgelegt habe. Weiters versichere ich, dass die von mir eingereichten Exemplare (ausgedruckt und elektronisch) identisch sind.
Datum: ………………………… Unterschrift: …………………………………………………
Abstract Die vorliegende Arbeit befasst sich mit der Diversität von Enzymen nitrifizierender Organismen sowie den damit verbundenen biotechnologischen Anwendungsmöglichkeiten und besteht aus zwei einander ergänzenden Teilen. Nitrifikation ist ein bedeutender Teil des Stickstoffzyklus und beschreibt die Oxidation von Ammonium zu Nitrit und anschließend zu Nitrat. Dabei wird Ammonium von Ammonium- oxidierenden Bakterien (AOB) oder Ammonium-oxidierenden Archaea (AOA) umgewandelt, während Nitrit-oxidierende Bakterien (NOB) Nitrit zu Nitrat verstoffwechseln. Beide Gruppen können jeweils nur ein Substrat (Ammonium oder Nitrit) oxidieren. Die funktionale Teilung der Nitrifikation in diese zwei Teilschritte galt lange als opinio communis, bis diese 2015 durch die Entdeckung von Comammox-Bakterien geändert werden musste. Comammox ist ein Akronym und steht für complete ammonia oxidizer; diese Bakterien können daher sowohl Ammonium zu Nitrit als auch Nitrit zu Nitrat oxidieren. Teil I der Arbeit beschäftigt sich mit zwei Proteinen (Nxr und MCO) eines Comammox Bakteriums (Candidatus Nitrospira inopinata) und einer Cyanase eines Ammonium- oxidierenden Archaeon (Nitrososphaera gargensis). Der Proteinkomplex Nitritoxidoreduktase (Nxr) besteht aus drei Untereinheiten und ist das Schlüsselenzym für die Oxidation von Nitrit zu Nitrat. Anders als bei anderen Nitrifizierern weist Nxr von Nitrospira Arten eine periplasmische statt einer zytoplasmischen Orientierung auf, wodurch eine Ansammlung toxischen Nitrits im Zytoplasma verhindert werden kann. Die Multicopperoxidase (MCO) von Ca. N. inopinata ist ein neuartiges Protein, dessen Funktion noch unbekannt ist. Ziel ist es, für beide Proteine einen Weg zu finden sie zu klonieren, zu exprimieren und anschließend aufzureinigen. Die Cyanase stammt von N. gargensis und katalysiert die Reaktion von Cyanat zu Carbamat, das anschließend zu Ammoniak und Kohlendioxid zerfällt. Im Vorfeld konnte gezeigt werden, dass die N. gargensis der erste bekannte Organismus ist, der Cyanat als einzige Energiequelle nutzen kann. Das Enzym konnte erfolgreich exprimiert und aufgereinigt werden. Das aufgereinigte Protein ist der Ausgangspunkt für weitere biophysikalische Analysen; mithilfe von Size Exclusion Multiangle Light Scattering (SEC-MALS) konnte festgestellt werden, dass das Enzym Cyanase als Oktamer vorliegt. Kinetische Analysen haben gezeigt, dass die Affinität der Cyanase von N. gargensis auf das Substrat Cyanat höher ist als bei anderen Organismen (z.B. E. coli). Teil II befasst sich mit dem möglichen Einsatz von Comammox Bakterien in modernen Abwasseranlagen. Dazu wird zuerst ein Überblick über die Hauptkläranlage Wien gegeben. Anschließend folgt eine Zusammenfassung von neuen Technologien basierend auf Anammox- Bakterien (anaerobic ammonia oxidation). Dabei wird die anaerobe Oxidation von Ammonium zu Nitrit mit der Reduktion von Nitrit zu Stickstoff (N2) gekoppelt. In der Abwassertechnologie wird der Anammox-Prozess mit der aeroben Oxidation von Ammonium zu Nitrit kombiniert. Zum Schluss wird ein Ausblick über mögliche Anwendungen von Comammox in der Hauptkläranlage Wien gegeben.
Abstract The present thesis deals with the diversity of enzymes of nitrifying organisms and the associated biotechnological applications. It consists of two complementary parts. Nitrification is an important part of the nitrogen cycle and describes the oxidation of ammonia to nitrite and subsequentially to nitrate. During this process, ammonia is converted by ammonia-oxidizing bacteria (AOB) or ammonia-oxidizing archaea (AOA), while nitrite- oxidizing bacteria (NOB) metabolize nitrite to nitrate. Both groups can only oxidize one substrate (ammonia or nitrite). The functional division of nitrification into these two sub-steps was long considered opinio communis, until this had to be changed in 2015 with the discovery of Comammox bacteria. Comammox is an acronym and stands for complete ammonia oxidizer; these bacteria can therefore oxidize ammonia via nitrite to nitrate. Part I of the work deals with two proteins (Nxr and MCO) of a Comammox bacterium (Candidatus Nitrospira inopinata), as well as with a cyanase of an ammonium oxidizing archaeon (Nitrososphaera gargensis). The protein complex nitrite oxidoreductase (Nxr) consists of three subunits and is the key enzyme for the oxidation of nitrite to nitrate. Unlike other nitrifiers, Nxr of Nitrospira exhibits periplasmic rather than cytoplasmic orientation, which can prevent accumulation of toxic nitrite in the cytoplasm. The multicopperoxidase (MCO) of Ca. N. inopinata is a novel protein whose function is still unknown. The goal is to find a way to clone, express and purify both proteins. The cyanase comes from N. gargensis and catalyzes the reaction of cyanate to carbamate, which then decomposes to ammonia and carbon dioxide. It could be shown beforehand that N. gargensis is the first known organism that can use cyanate as its only energy source. The enzyme was successfully expressed and purified. The purified protein is the starting point for further biophysical analyses; with the help of size exclusion multiangle light scattering (SEC- MALS) it could be determined that the enzyme cyanase is present as an octamer. Kinetic analyses have shown that the affinity of the cyanase of N. gargensis to substrate cyanate is higher than in other organisms (e.g. E. coli). Part II deals with the possible use of Comammox bacteria in modern wastewater plants. For this purpose, an overview of Vienna’s main wastewater treatment plant is given first. This is followed by a summary of new technologies based on Anammox (anaerobic ammonia oxidation) bacteria. The anaerobic oxidation of ammonia to nitrite is coupled with the reduction of nitrite to nitrogen (N2). In wastewater technology the anammox process is combined with the aerobic oxidation of ammonia to nitrite. Finally, an outlook on possible applications of Comammox in Vienna’s main wastewater treatment plant is given.
Acknowledgments First and foremost, I would like to express my gratitude to my supervisor Univ.-Prof. Dr. Dipl. Ing. Kristina Djinovic-Carugo, who has given me the opportunity to do this master project in her lab. With her valuable feedback she has contributed substantially to this thesis. Dr. Andreas Franz has sparked my interest in wastewater treatment and opened my eyes to this fascinating world. I would like to thank him not only for this, but also for his assistance during the writing. I want to express my deepest gratitude to Mag. Dr. Georg Mlynek, who has been my mentor and has always had an open ear for all my problems. Thanks is also due to Mag. Julius Kostan, PhD and Antonio Sponga, MSc for their help in the lab. Dr. Chris Sedlacek from the department for microbial ecology has helped me a lot with the cyanase, and I would like to thank him for that. Lastly, I would like to thank all my colleagues from the Djinovic Lab for all their support, help and shared moments, which I will always cherish. Table of contents
PART I 1 Introduction ...... 3 1.1 Nitrification ...... 3 1.2 Candidatus Nitrospira inopinata ...... 4 1.2.1 Nitrite oxidoreductase (Nxr) of Ca. N. inopinata ...... 5 1.2.2 Multicopperoxidase (MCO) from Ca. N. inopinata ...... 7 1.3 Nitrososphaera gargensis ...... 7 1.3.1 Cyanase from N. gargensis ...... 8 1.4 Research questions...... 10 2 Materials and methods ...... 12 2.1 Cloning ...... 12 2.2 Small-scale expression ...... 14 2.3 Large scale expression and purification ...... 16 2.4 SEC-MALS ...... 18 2.5 CynS activity assay ...... 18 3 Results ...... 19 3.1 Cloning ...... 19 3.2 Small scale expression ...... 19 3.3 Large scale expression and purification ...... 22 3.4 SEC-MALS ...... 24 3.5 CynS activity assay ...... 25 4 Discussion and outlook ...... 29 4.1 Cloning ...... 29 4.2 Expression ...... 29 4.3 CynS activity assay ...... 29
PART II
5 Utilization of comammox in wastewater treatment ...... 31 5.1 The role of nitrification and denitrification in wastewater treatment ...... 31 5.2 Wastewater treatment parameters ...... 33 5.3 Wastewater treatment explained on the concept employed on the Vienna Main Wastewater Treatment Plant (VMWWTP) ...... 34 5.4 Anammox and Deammonification...... 39 5.5 Deammonification process technologies ...... 41
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5.5.1 ANAMMOX® Granulated Sludge Reactor ...... 41 5.5.2 DEMON® Sequencing batch reactor ...... 42 5.5.3 Moving Bed Biofilm reactors (MBBR) ...... 44 5.6 Comammox and WWTP ...... 46 6 Abbreviations ...... 50 7 List of figures ...... 51 8 List of tables...... 52 9 References ...... 52
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PART I
1 Introduction 1.1 Nitrification Nitrification and denitrification, important parts of the so-called nitrogen cycle (fig.1), also play key roles in the biological purification of modern wastewater treatment plants (WWTP). - - In WWTP nitrogen can be found in various forms: ammonia (NH3), nitrite (NO2 ), nitrate (NO3
) or organic compounds, such as urea (CH4N2O). The efficient removal of these compounds is vital for the environment as the treated wastewater from the plants is directed into our rivers, causing eutrophication which finally can lead to oxygen depletion and death of aquatic life.
The nitrogen cycle is a complex cycle combining and linking various processes together. Although nitrogen is needed by all living organisms, its bioavailability can be problematic and therefore become a growth-limiting factor (1)-(2). The most abundant form of nitrogen on the planet is atmospheric nitrogen gas (N2). Nitrogen fixation marks the beginning of the nitrogen cycle by the conversion of nitrogen gas into ammonia (3). On an industrial scale, this is achieved by the Haber-Bosch process.
Fig. 1: Nitrogen compounds and their relationships among each other. Nitrogen fixation is followed by ammonification from organic nitrogen in animals or plants. The resulting ammonium is then oxidized to nitrite and nitrate. Figure modified after: (4).
Nitrification is the biological oxidation of ammonia via nitrite [1] to nitrate [2]. Sergei Winogradsky described nitrification as a two-step process done by two different species:
3 ammonium oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) on the one hand, and nitrite oxidizing bacteria (NOB) on the other (5).
One of the most common genera of the AOB’s are Nitrosomonas. They are chemoautolithotrophic organisms and are abundant in different habitats such as soils, marine, freshwater systems, and wastewater treatment plants (WWTP). AOBs use two key enzymes for the oxidation of ammonia to nitrite:
First, the membrane-bound enzyme ammonia monooxygenase (AMO) oxidizes ammonia to hydroxylamine (H3NO), which is subsequently further oxidized by periplasmic hydroxylamine dehydrogenase (6) to nitrite (7). The conversion of ammonia into nitrite is the rate-limiting step in the whole nitrification process. In 2005 the discovery of ammonia oxidizing archaea (AOA) was widely reported. Their oxidation pathway from ammonia to nitrite, however, is still not yet fully understood, since no hydroxylamine oxidase (6) homologue has ever been found in their genome (8).
- + [1] NH3 + 1.5 O2 NO2 + H2O + 2H
- - [2] NO2 + 0.5 O2 NO3
The second step – oxidation of nitrite into nitrate – is done by chemolithoautotrophic nitrite oxidizing bacteria (NOB). Nitrobacter and Nitrospira are prominent representatives of this group.
The microorganisms involved in the two steps process are not closely related. None of them can oxidize both substrates, which leads to cross-feeding interaction between ammonia and nitrite oxidizers (9). This also explains why toxic nitrite is rarely accumulated under oxic conditions (10). The key enzyme for nitrite oxidation is nitrite oxidoreductase (Nxr). The membrane-associated protein complex consists of three subunits (alpha, beta, and gamma); NxrA is the nitrite binding and catalytic site, NxrB channels two electrons per oxidized nitrite from NxrA via four iron-sulfur-clusters to NxrC (11).
1.2 Candidatus Nitrospira inopinata As described above, nitrification had been thought to be a two-step process performed by two distinct groups of microorganisms. From a thermodynamical point of view, this functional separation was surprising, since incomplete nitrification yields less energy (ΔG°’ = -275 kJ mol- 1 -1 - -1 NH3 and ΔG°’ = -74 kJ mol NO2 ) compared to complete nitrification (ΔG°’ = -349 kJ mol
NH3) (9). Therefore, it had been surmised that a hypothetical organism, capable of complete nitrification, must exist. This organism was called comammox (complete ammonia oxidizer). Based on kinetic theory, however, cross-feeding ammonia and nitrite oxidizers would outcompete comammox in most environments, except in substrate limited environments such as biofilms (12).
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In 2015 comammox organisms could be identified for the first time (9), (13). Three Nitrospira species (Ca. N. inopinata, Ca. N. nitrosa and Ca. N. nitrificans) were identified as comammox.
Ca. N. inopinata was found in a microbial biofilm from a pipe, in which hot water (56°C) was constantly flowing. The culture was incubated at 46°C in mineral media with ammonium. Fluorescence in situ hybridization confirmed that the culture contained no other nitrifiers except Nitrospira. Furthermore, no bacterial or archaeal genes of AMO and 16S rRNA genes of AOA could be detected by PCR. The genome was sequenced and it was confirmed that Ca. N. inopinata possesses genes for nitrite oxidation (Nxr) as well as ammonia oxidation (AMO and HAO) (9).
1.2.1 Nitrite oxidoreductase (Nxr) of Ca. N. inopinata Nitrite oxidoreductase is the key enzyme of nitrite-oxidizing bacteria (NOB) and catalyzes the - - oxidation of nitrite into nitrate (NO2 to NO3 ). NOBs are a phylogenetically diverse group. Its representatives are ubiquitous in nature as well as in engineered systems, like wastewater treatment plants, but hard to cultivate. Thus, research on them had not been pursued for a long time. At the moment, NOBs of seven genera in four bacterial phyla are known (14). It turns out that the diversity of NOBs is not only due to the vast number of family members but also due to the composition of the Nxr complex. It differs in composition of the respiratory chain, as well as in the pathway used for autotrophic carbon fixation (15).
First studies on NOBs have focused on Nitrobacter, which belong to the class of Alphaproteobacteria (11). Depending on the purification method, their Nxr consists of two or three subunits: NxrA (α), NxrB (β) and NxrC (γ) with the stoichiometry of α2β2γ1. The protein complex is located at the intracytoplasmic membrane and the α-subunit faces in the cytoplasm (fig. 2B). NxrA binds the substrate and has a molybdenum cofactor in the catalytic - site. Per oxidized NO2 two electrons are shuttled from subunit α to γ or directly to the electron transport chain by the iron-sulfur containing subunit β. The cytoplasmic position results in the - - necessity of a nitrite/nitrate transporter, which shuffles NO2 and NO3 across the inner - membrane (16). Nxr not only catalyzes oxidation from nitrite to nitrate but also reduces NO3 with electrons from organic compounds (17).
Nitrospira members represent their own bacterial phylum and are the most diverse group within nitrite oxidizing bacteria. Unlike other nitrifiers, Nitrospira lack an intracytoplasmic membrane. Nitrospira moscoviensis had been the first representative of this group, which could be studied in a pure culture. Their Nxr is also a membrane bound protein complex (15), which consists of at least two subunits (NxrA and NxrB), similar to Nitrobacter. However, NxrA and NxrB are not located in the cytoplasm but in the periplasm (Fig. 2A). With this location the accumulation of toxic nitrite in the cytoplasm is avoided (18). This extracytoplasmic oxidation of nitrite leads to an enlarged periplasmic space in Nitrospira, as well as the formation of a proton gradient without the need for an energy-dependent permease system for nitrite (19).
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Fig. 2: Nitrite oxidoreductase types. (A): Periplasmic Nxr leads to an enlarged periplasmic space in Nitrospira species and avoids accumulation of toxic nitrite in the cytoplasm. (B): Cytoplasmic Nxr results in nitrite/nitrate transporter to shuffle NO2- and NO3- across the inner membrane. Figure modified after: (14).
Further research with Candidatus Nitrospira defluvii confirmed the periplasmic orientation of Nxr (fig 2A). NxrA also contains a N-terminal twin arginine signaling peptide for transporting folded proteins via the twin-arginine translocation (20) pathway (15). This transportation system does not rely on ATP as energy source, but depends on the proton motive force (21). A N-terminal signal peptide, consisting of a polar amino domain, hydrophobic core and carboxyl domain, is recognized by the Tat machinery and activates the secretion via this pathway. Although NxrB does not have a Tat-signal peptide, it is thought to hitchhike together with subunit α into the periplasm (15). Similar to NxrB in Nitrobacter, four iron-sulfur clusters could be identified.
Subunits α and β do not contain any transmembrane helices, which means that they are dependent on other membrane associated units. NxrA shows high similarity to the type II dimethyl sulfoxide (DMSO) reductase family, which contain an additional subunit γ (15). This subunit anchors the complex in the membrane and channels electrons from subunit β via one or two hemes to the electron transport chain (22).
The genome of Ca. N. inopinata contains one copy of the NxrA and NxrB subunits, as well as four copies of NxrC (9). This is in contrast to other Nitrospira spieces, which possess two to five gene copies of the NxrA and NxrB (15) subunits.
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- Unlike Nitrobacter (23), Nitrospira shows high affinity (low Km) to the substrate NO2 (15). Combined with the fact that high nitrite accumulation is not very common in nature, this may have led to the wide distribution of Nitrospira.
1.2.2 Multicopperoxidase (MCO) from Ca. N. inopinata Multicopperoxidase (MCO) from Ca. N. inopinata is a novel protein and little is known about its role in the nitrification process. It was proposed, however, that MCO is important for ammonia oxidation in AOAs (fig. 3). AOBs and AOAs differ in their oxidization pathways. The latter, for example, lack the enzyme HAO, which oxidizes hydroxylamine (NH2OH) to nitrite - (NO2 ). This conversion is the result of a reaction between NH2OH, NO and H2O and catalyzed by a Cu-containing enzyme. MCO could be the counterpart to bacterial HAO or a part of the archaeal hydroxylamine ubiquinone redox module (HURM) (24) (25).
Fig. 3: Proposed ammonia oxidation pathway for AOAs. Instead of a hydroxylamine dehydrogenase (6), the so-called - hydroxylamine ubiquinone redox module converts NH2OH into NO2 . This enzyme contains copper and MCO as a copper containing metalloenzyme might serve as Cu-containing enzyme. Figure modified after: (24).
One theory is that MCO together with NO play a significant role in the metabolism of Ca. N. inopinata and could lead to a new understanding of the nitrogen cycle. Structural analysis of the 186 kDa enzyme with 6 copper-centers would provide a better understanding of its function.
1.3 Nitrososphaera gargensis N. gargensis are chemoautolithotroph ammonia-oxidizing archaea (AOA). They belong to the phylum Thaumarchaeota and, together with Nitrosopumilus, are the most abundant members
7 within this group. One of their closest relatives is Nitrosophaera viennensis (26), with which they share 86.3 % sequence identity (27). N. gargensis is a moderately thermophilic organism that grows optimally at 46°C (28).
Even though they have a gene set for ammonia oxidation and carbon dioxide fixation, they are not solely dependent on free ammonia as substrate. Their genome shows genes for urease and cyanase, which means they can produce ammonia from either urea or cyanate. Especially the presence of a cyanase is unique, as N. gargensis is the only known bacterial or archaeal ammonia oxidizer or member of the Thaumarchaeota phylum respectively, that encodes this enzyme. Since N. gargensis shares most central metabolic pathways with other members of this phylum, it is unlikely that it is the only representative, which needs cyanase for detoxification of intracellular produced cyanate. It was rather shown, that N. gargensis is the first known organism that can use cyanate as its sole source of energy and reductant for growth (29).
Interestingly, the cyanase gene is located in close proximity to the formate/nitrite transporter, which would indicate the presence of a cyanate transporter (27). Furthermore, “the colocalization of the cyanate uptake and conversion module suggests that the respective genes can easily be transferred together and that N. gargensis has exchanged these genes with nitrifiers sharing a similar niche” (27).
In contrast to other ammonia-oxidizers, all known nitrite-oxidizers (such as Nitrospira) encode genes for cyanase. This is beneficial for multiple reasons (29):
Cyanate is formed during carbamoyl phosphate metabolism and urea formation. Many NOBs, however, lack enzymes for degradation of internally produced urea and therefore also produce more cyanate. They need cyanases to convert the intracellularly accumulated cyanate to ammonium and carbon dioxide. The nitrite transporters of NOBs are also capable of transporting cyanate, which probably leads to an increased uptake of cyanate from the environment. Ammonia and nitrite oxidizers live in cross-feeding communities, since AOM cannot convert cyanate but NOBs can. Therefore, NOBs use cyanases to detoxify their produced and absorbed cyanate and in doing so they also produce ammonia, which serves as an energy source and reductant for AOMs. These, in turn, produce nitrite, which is needed by NOBs.
1.3.1 Cyanase from N. gargensis Cyanase (also known as cyanate hydratase or cyanate lyase) catalyzes the reaction of cyanate to carbamate [3], which subsequently decomposes to ammonia and carbon dioxide [4]. This reaction is bicarbonate-dependent (30).
- - + - [3] NCO + HCO3 + H H2NCOO + CO2
- + [4] H2NCOO + H NH3 + CO2 8
Cyanate can be spontaneously formed by isomerization in aqueous solutions (31). In the cell cyanate is toxic, since it reacts with the nucleophilic groups in proteins and therefore alters their structure. Most organisms use cyanases for detoxification (32). Cyanases occur in a variety of organisms, such as bacteria (33)-(34), proteobacteria (35), cyanobacteria (36), fungi (37), plants (38) and archea (27).
The first characterized cyanase was from E. coli. The crystal structure (fig. 4) reveals that the active enzyme forms a pentameric structure of dimers (homodecamer; monomeric subunit 17 kDa). The crystal structure of cyanase from Serratia proteamaculans shows the same composition as the cyanase of E. coli (decamer of five dimers) (39). The dimers are connected by an intermolecular cysteine bridge (40). The interface of the five dimers carry a unique set of amino acid residues, which enable the enzyme to bind and catalyze the reaction between cyanate and bicarbonate (41). Three catalytic residues (Arg96, Glu99, Ser122) of the cyanase from E. coli are even preserved in cyanases from proteobacteria and fungi (38). Ser122 is important for substrate binding, while the guanidinium group of Arg96 stabilizes the negatively charged substrates (cyanate and/or bicarbonate). An activated water molecule bound to Glu99 protonates the cyanate nitrogen. This reaction leads to a more electrophilic carbon atom of cyanate, and due to a nucleophilic attack by the bicarbonate carboxylate oxygen a dianion intermediate is formed, which subsequently decarboxylates to carbon dioxide and carbamate (41).
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(a) (b)
(c)
Fig. 4: (a): Crystal structure of cyanase from E. coli (PDB: 1DW9). The enzyme is formed by dimeric and decameric arrangement of subunits. The active site is located at the interface of the dimers, which is indicated by bound oxalate (purple) as a weak inhibitor. (b) Two monomers forming a dimer are colored in green and violet. (c): Representation of the active site with the involved catalytic residues and bound oxalate. Figure modified after: (41).
1.4 Research questions Part I is part of an ongoing research project. Escherichia coli is a major expression host for recombinant industrial scale protein production (42). The available genetic toolbox, easy and low-cost handling and fast turnover rates provide the foundation of its popularity. To fully exploit the potential of E. coli, factors influencing the expression of heterologously expressed proteins have to be optimized for every single target.
Even though nitrite-oxidoreductases (Nxr) are well-known proteins, Nxr of a comammox bacterium is being here studied for the very first time. Since Nxr is an intricate protein
10 complex, the goal of this study is to find conditions for a successful co-expression of NxrA and NxrB with the respective chaperone in E. coli.
MCO from Ca. N. inopinata is a novel protein and little is yet known about it. As for the NxrAB complex, the main goal is to discover a way to periplasmically express this protein.
The third protein is a cyanase from N. gargensis. This is the first archael cyanase being studied. The goal is to clone, express and biochemically characterize this protein in order to compare it to other known cyanases. This will be the preparative work for crystallizing the protein and to further understand the only microorganism that can use cyanate as its sole source of energy and reductant.
Part II focuses on the industrial utilization of comammox bacteria in modern wastewater treatment plants. Known nitrogen removal technologies will be compared with each other and possible applications in the Vienna Main Wastewater Treatment Plant will be discussed.
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2 Materials and methods 2.1 Cloning Cloning of the individual constructs was done using genomic DNA from Ca. N. inopinata, Nitrospira moscoviensis and Nitrososphaera gargensis or codon optimized DNA for expression in E. coli.
The gene of interest (GOI) was cloned in vectors comprising different affinity tags and antibiotic resistance. (tab. 1-4).
Nxr Constructs
Tab. 1: Overview of the cloned Nxr-constructs
Nxr Constructs GOI Vector Affinity Tags Resistance Origin Description NxrB p3NOS_CH_ccdB N-strep, C-6H Kanamycin N. inopinata Genomic DNA NxrB p3NOS_CH_ccdB N-strep, C-6H Kanamycin N. moscoviensis Genomic DNA C-6H (subunit NxrAB pET21+ Ampicillin N. inopinata codon optimized B) C-6H (subunit NxrAB p1 Ampicillin N. inopinata codon optimized B)
Nxr Chaperone Constructs
Tab. 2: Overview of the cloned Nxr chaperone-constructs
Nxr Chaperone Constructs GOI Vector Affinity Tags Resistance Origin Description Chap p3NH N-6H Kanamycin N. inopinata native Chap pETM22 N-trx, N-6H Kanamycin N. inopinata native Chap pETM33 N-6H, N-GST Kanamycin N. inopinata native Chap pETM44 N-6H, N-MBP Kanamycin N. inopinata native Chap pCOLADuet-1 - Kanamycin N. inopinata codon optimized Chap pCOLADuet-1 C-6H Kanamycin N. inopinata codon optimized Chap p3NH N-6H Kanamycin N. inopinata codon optimized Chap pETM22 N-trx, N-6H Kanamycin N. inopinata codon optimized Chap pETM33 N-6H, N-GST Kanamycin N. inopinata codon optimized Chap pETM44 N-6H, N-MBP Kanamycin N. inopinata codon optimized
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MCO constructs
Tab. 3: Overview of the cloned MCO-constructs
MCO Constructs GOI Vector Affinity Tags Resistance Origin Description MCO p3NOS_CH_ccdB N-strep, C-6H Kanamycin N. inopinata Genomic DNA MCO p3NOS_CH_ccdB N-strep, C-6H Kanamycin N. moscoviensis Genomic DNA MCO p3NH N-6H Kanamycin N. inopinata codon optimized
CynS-Constructs
Tab. 4: Overview of the cloned cynS-constructs
CynS Constructs GOI Vector Affinity Tags Resistance Origin Description cynS p3NOS_CH_ccdB N-strep Kanamycin N. gargensis Genomic DNA cynS p3NH N-6H Kanamycin N. gargensis Genomic DNA cynS pETM22 N-trx, N-6H Kanamycin N. gargensis Genomic DNA cynS pETM33 N-6H, N-GST Kanamycin N. gargensis Genomic DNA cynS pETM44 N-6H, N-MBP Kanamycin N. gargensis Genomic DNA
All constructs were cloned using Gibson assembly method (43). A 50 µL polymerase chain reaction (PCR) consists of 20 ng template DNA, 1 µM forward and reverse primer, 23 µL ddH2O 25 µl 2x Phusion High Fidelity Master Mix (ThermoFisher Scientific). DNA amplification was done, using the following program (tab. 5).
Tab. 5: PCR program
PCR Program Step Temperature [°C] Time Number of Cycles Initial denaturation 98 15 s 1 Denaturation 98 15 s Annealing 55-65 15 s 25 Elongation 72 15 s/kb Final elongation 72 10 min 1 Hold 10 1
Amplification was checked on a 1 % agarose gel and the product purified using the GeneJET PCR purification kit (ThermoFisher Scientific To digest the parental DNA, the PCR product was incubated with 10 U DpnI and 1x Tango buffer (both ThermoFisher Scientific) at 37°C for 1h.
After another DNA purification step with GeneJET PCR purification kit (ThermoFisher Scientific), the DNA fragments were assembled using Gibson. Therefore, 2.5 µl of vector and insert are mixed in a 1:3 molarity ratio together with 7.5 µl 4/3 Gibson assembly master mix
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(5x IT buffer, T5 exonuclease, Phusion HF DNA polymerase, Taq DNA ligase, nuclease-free water). The reaction was carried out at 50°C for 1h.
5 µl of the reaction mix was transformed into TOP10 competent cells using heat shock method and plated on agar plates with the desired antibiotic resistance. After overnight growth at 37°C, single colonies were picked, DNA was isolated with GeneJET Plasmid Miniprep Kit (ThermoFisher Scientific) and sent for sequencing to Eurofins to confirm the correct DNA sequence.
2.2 Small-scale expression
The following constructs (tab. 6-9) are expressed in small-scale to test protein expression and solubility. For all small-scale expression experiments, the following protocol was used: Constructs were transformed into desired E. coli strains using heat shock method and plated on agar plates with desired antibiotics. A preculture was prepared by suspending cells from the plate in LB medium with antibiotics and by letting them grow over night at 37°C. 5 ml of LB, ZY or YT media with antibiotics were inoculated with 100 µl preculture. Cells grew until an
OD600 of 0.8 was reached at 37°C. Cells were induced with 0.2 or 0.5 mM IPTG (except cells in ZY medium) and grown either at 37°C or at 18°C after induction over night. At the moment of induction, the NxrB-cultures were supplemented with different additives (200 µl iron-sulfur mix for NxrB-cultures, 50 µl 2 mM CuCl2 for MCO cultures). The iron-sulfur mix consisted of 2 mM ferric citrate (C6H5FeO7), 2 mM iron sulfate heptahydrate (FeSO4 · 7 H2O), 2 mM ferric ammonium citrate and 100 mM cysteine (44).
Cells are harvested by centrifugation at 3202 rcf for 20 min. The supernatant was discarded and the pellet stored for at least 1h at -80°C. The pellet was first suspended in 600 µl of 1x PBS, 1x BugBuster and 0.25 µl DNAseI / ml (stock 10 mg/ml) and then incubated for 20 min at 20°C while shaking at 1300 rpm. To separate the soluble and insoluble fractions, the suspension was centrifuged for 20 min at 3202 rcf.
The soluble fraction was purified with the following protocol: 200 µl of 4xNi Sepharose 6 FF beads were added to lysate and incubated for 15 min at 20°C. The lysate with added Ni-beads was transferred to a 50 µm hydrophilized receiver plate (Macherey-Nagel). Before elution with 150 µl 1x PBS and 500 mM imidazole, the plate was washed twice with 1 ml 1x PBS.
To purify the periplasm the following protocol was used: 1 g of pellet was resuspended in 5 ml lysis buffer (100 mM HEPES, pH 8.0, 500 mM NaCl, 10% glycerol). Then 1 mM of EDTA and 0.5 mg/ml lysozyme were added and incubated for 1 h at 20°C. The periplasmic fraction was separated by centrifuging for 20 min at 3202 rcf.
14
Tab. 6: Small-scale expressed Nxr constructs
Expression Aerobic/ IPTG Construct Origin Description T [°C] Media kDa System Anaerobic [mM] BL21, C41, 37 on, NxrB_p3NOS N. 0.2, native BL21pLysS, 37/18 LB, ZY aerobic 54.1 _CH_ccdB inopinata 0.5 R2pLysS on
N. BL21, C41, 37 on, NxrB_p3NOS 0.2, moscoviensi native BL21pLysS, 37/18 LB, ZY aerobic 54.1 _CH_ccdB 0.5 s R2pLysS on NxrAB_pET21 N. codon BL21, 37/18 LB, ZY, aerobic, 0.2, 131.7, 50.6 + inopinata optimized R2pLysS on YT anaerobic 0.5 NxrAB_pET21 + & N. codon BL21, 37/18 LB, ZY, aerobic, 0.2, 131.7, 50.6 Chap_pCOLA inopinata optimized R2pLysS on YT anaerobic 0.5 Duet-1 NxrAB_pET21 + & N. codon BL21, 37/18 LB, ZY, aerobic, 0.2, 131.7, 50.6 Chap_6H_pC inopinata optimized R2pLysS on YT anaerobic 0.5 OLADuet-1
Tab 7: Small-scale expressed Nxr chaperone-constructs
Expression Aerobic/ IPTG Construct Origin Description T [°C] Media kDa system Anaerobic [mM] Chap_pCOLA N. codon BL21, 37/18 LB, ZY, aerobic, 0.2, 36.2 Duet-1 inopinata optimized R2pLysS on YT anaerobic 0.5 Chap_6H_pC N. codon BL21, 37/18 LB, ZY, aerobic, 0.2, 37 OLADuet-1 inopinata optimized R2pLysS on YT anaerobic 0.5 BL21, N. codon 37/18 aerobic, 0.2, Chap_p3NH BL21pLyS, LB, ZY 38.3 inopinata optimized on anaerobic 0.5 R2pLysS BL21, N. 37/18 aerobic, 0.2, Chap_p3NH native BL21pLyS, LB, ZY 38.3 inopinata on anaerobic 0.5 R2pLysS BL21, N. codon 37/18 aerobic, 0.2, Chap_petM22 BL21pLyS, LB, ZY 50.5 inopinata optimized on anaerobic 0.5 R2pLysS BL21, aerobic, N. 37/18 0.2, Chap_petM22 native BL21pLyS, LB, ZY anaerobic 50.5 inopinata on 0.5 R2pLysS BL21, aerobic, N. codon 37/18 0.2, Chap_petM33 BL21pLyS, LB, ZY anaerobic 64.4 inopinata optimized on 0.5 R2pLysS BL21, aerobic, N. 37/18 0.2, Chap_petM33 native BL21pLyS, LB, ZY anaerobic 64.4 inopinata on 0.5 R2pLysS BL21, aerobic, N. codon 37/18 0.2, Chap_petM44 BL21pLyS, LB, ZY anaerobic 78.8 inopinata optimized on 0.5 R2pLysS BL21, aerobic, N. 37/18 0.2, Chap_petM44 native BL21pLyS, LB, ZY anaerobic 78.8 inopinata on 0.5 R2pLysS 15
Tab. 8: Small-scale expressed MCO-constructs
Expression Aerobic/ IPTG Construct Origin Description T [°C] Media kDa System Anaerobic [mM] BL21, C41, 37 on, MCO_p3NOS N. 0.2, native BL21pLysS, 37/18 LB, ZY aerobic 176 _CH_ccdB inopinata 0.5 R2pLysS on
N. BL21, C41, 37 on, MCO_p3NOS 0.2, moscoviensi native BL21pLysS, 37/18 LB, ZY aerobic 176 _CH_ccdB 0.5 s R2pLysS on BL21, N. codon 37/18 aerobic, 0.2, MCO_p3NH BL21pLysS, LB, ZY 174 inopinata optimized on anaerobic 0.5 R2pLysS
Tab 9: Small-scale expressed cynS-constructs
Expression Aerobic/ IPTG Construct Origin Description T [°C] Media kDa System Anaerobic [mM] BL21, C41, 37 on, cynS_p3NOS_ N. 0.2, native BL21pLysS, 37/18 LB, ZY aerobic 22.5 CH_ccdB gargensis 0.5 R2pLysS on BL21, aerobic, N. 37/18 LB, ZY, 0.2, cynS_p3NH native BL21pLysS, anaerobic 20.4 gargensis on YT 0.5 R2pLysS BL21, aerobic, N. 37/18 LB, ZY, 0.2, cynS_pETM22 native BL21pLysS, anaerobic 32.6 gargensis on YT 0.5 R2pLysS
2.3 Large scale expression and purification Nxr-Constructs Plasmids NxrB_p3NOS_CH_ccdB from Ca. N. inopinata and N. moscoviensis were transformed into BL21 by heat shock and plated on agar plates with 50 µg/ml kanamycin. 3 l of LB media were inoculated with 30 ml overnight culture. Cells grew at 37°C until they reached OD600 of 0.8. At that point they were induced with 0.5 µM IPTG and temperature was lowered to 18°C. Additionally, cells were supplied with 120 ml iron sulfur mix. Cells were harvested by centrifuging at 3202 rcf for 20 min in a HITACHI centrifuge.
The pellet was resuspended in 20 mM TRIS-HCl, 300 mM NaCl, 10 µM CuCl2 (pH 7.0) and 50 µL DNAseI (stock 10 mg/ml), homogenized, sonicated (twice 3x3 min, 50% amplitude) and centrifuged at 4°C and 3202 rcf for 20 min in a HITACHI centrifuge. For the purification an Aekta pure from GE healthcare was used. The supernatant was loaded on a 5 ml StrepTrap HP
(GE Healthcare) equilibrated with Buffer A: 20 mM TRIS-HCl, 300 mM NaCl, 10 µM CuCl2 (pH
7.0). Buffer B: 20 mM TRIS-HCl, 300 mM NaCl, 10 µM CuCl2, 2.5 mM desthiobiotin (pH 7.0) was used for elution 16
MCO-Constructs The procedure of large-scale expression and purification of MCO_p3NOS_CH_ccdB from Ca. N. inopinata and N. moscoviensis was the same as for the Nxr-constructs with the difference that upon induction 20 µM CuCl2 instead of the sulfur mix was added.
CynS-Constructs For the expression the construct cynS_pETM22 was used. The plasmid was transformed into R2plysS cells by heat shock and plated on agar plates with 50 µg/ml Kanamycin. 3 l of ZY media were inoculated with 30 ml overnight culture. Cells grew until an OD600 of 1.16 at 37°C and then the temperature was lowered to 18°C. Cells were harvested by centrifugation at 3202 rcf for 20 min in a HITACHI centrifuge.
The pellet was resuspended in 1x PBS, 20 mM imidazol, pH 7.4 and supplemented with 50 µL DNAseI (stock 10 mg/ml), homogenized, sonicated (2x3 min, 50% amplitude) and centrifuged at 4°C and 3202 rcf for 20 min in a HITACHI centrifuge. For the purification an Aekta pure from GE healthcare was used. The supernatant was loaded on a 5 ml HisTrap Crude FF (GE Healthcare) and processed with the method shown in tab. 10. Buffer A: 1x PBS, 20 mM imidazol, pH 7.4. Buffer B 1x PBS, 500 mM imidazol, pH 7.4. After the His-Trap the 6H-tag was cleaved off over night with 3C protease (1:50, w/w) whilst dialyzed against 1xPBS. A reverse His-trap was performed to separate the cleaved from the uncleaved protein.
Tab. 10: Method for HisTrap (CV: column volume, % B: percentage of buffer B used). With a HisTrap histidine-tagged proteins are purified by immobilized metal ion affinity chromatography (IMAC).
HisTrap CV % B Equilibration 8 0 Sample application Column Wash 6 4
Elution 1st step 5 46 Elution 2nd step 5 100 Equilibration 3 0
The peak fraction was concentrated and loaded on a Superdex HiLoad S200 26/600 (GE Healthcare) and separated using an isocratic gradient for 1.2 CV in 1x PBS. Fractions of different peaks were pooled separately. The pool containing octameric cyanase was concentrated to 9.4 mg/ml and stored at -80°C.
17
2.4 SEC-MALS An Agilent 1260 Infinity HPLC system, equipped with a miniDAWN® MALS detector, and refractive index detector RI-101 was used to perform the experiment.
After equilibration over night with the SEC-MALS buffer (50 mM TRIS-HCl, 150 mM NaCl, pH: 7.5), 50 µL (8.3 mg/ml) of the main peak (orange, fig. 10) and 100 µL of the pooled fractions of the second peak (green, fig. 10) cynS were loaded on a Superdex increase S200 10/300 GL (GE Healthcare) column.
2.5 CynS activity assay CynS activity was determined by the conversion of cyanate to ammonia. The activity buffer consisted of 50 mM potassium phosphate buffer (pH 7.7) and 3 mM sodium bicarbonate. Both the substrate (potassium cyanate) and the enzyme (cynS) were suspended in the activity buffer. The reaction was initialized by adding cynS and stopped by the addition of Nessler reagent after 1-10 min at RT. The Nessler reagent forms a reddish-brown complex [Hg2N] with ammonia, the absorbance of which can be measured at 405 nm.
To determine enzyme kinetics the double reciprocal plot (Lineweaver Burk) was used. The substrate concentration varied (50, 60, 75, 90, 100, 125 µM), while the enzyme concentration was constant at 500 nM. Every point was measured three times.
As inhibitor sodium azide (NaN3) was dissolved in the activity buffer. Firstly, OD405 was measured from reactions of 100 µM KOCN with different inhibitor concentrations (10, 50, 100,
200 and 500 µM NaN3) after 10 min. Secondly, the type of inhibition of NaN3 on cyanase was determined by keeping the inhibitor concentration constant (50 µM) and varying the substrate concentration (50, 60, 75, 90, 100, 125 µM).
For the standard curve different concentrations (100, 200, 250, 300, 400, 500, 600 and 700
µM) of NH4Cl were suspended in the activity buffer. After the addition of the Nessler reagent the absorbance at 405 nm was measured.
18
3 Results 3.1 Cloning Cloning of all constructs described above was successful.
3.2 Small scale expression Nxr-Constructs Construct NxrB_p3NOS_CH_ccdB showed overexpression in the condition shown in tab. 11. This condition was used for large scale expression and purification. NxrB, however, co- expressed and co-purified with the chaperone GroEL (see chapter 3.3 for details). The codon optimized construct NxrAB_pET21+ did not show any overexpression in the tested conditions.
Tab. 11: Overview of small-scale expressed Nxr-constructs. All constructs originate from N. inopinata except constructs marked with an asterix (*), which originate from N. moscoviensis.
Nxr constructs Construct Affinity Tags Conditions of Expressed Protein NxrB_p3NOS_CH_ccdB N-strep, C-6H BL21, LB, 0.5 µM IPTG, 37°/18°C on
native native DNA NxrB_p3NOS_CH_ccdB* N-strep, C-6H BL21, LB, 0.5 µM IPTG, 37°/18°C on
NxrAB_pET21+ C-6H (subunit B) no expression codon optimized
The codon optimized NxrAB_pet21+ was also co-expressed with the corresponding chaperone (Chap-pCOLADuet-1). None of the tried conditions lead to an overexpressed protein (tab. 12)
Tab. 12: Overview of small scale co- expressed NXR and chaperone constructs. All constructs originate from N. inopinata.
Co-expressed Nxr and Nxr chaperone-constructs Conditions of Construct 1 Affinity Tags Construct 2 Affinity Tags Expressed Protein
Chap_pCOLADuet-1 - NxrAB_pET21+ C-6H (subunit B) no expression
codon optimized Chap_pCOLADuet-1 C-6H NxrAB_pET21+ C-6H (subunit B) no expression
After the screening of the codon optimized construct Chap-pCOLADuet-1, which showed no overexpression, the same gene of interest was cloned into different vectors (pET33, pETM44) with solubility tags (GST and MBP) (fig. 5, tab. 13). The SDS-PAGES showed that the tags increased protein expression
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Fig. 5: SDS-PAGE of Chap_pETM33 and Chap_pETM44. In both cases expressing the GOI with a solubility tag (GST and MBP) increased expression of soluble protein.
Tab. 13: Overview of small-scale expressed Nxr chaperone-constructs. All constructs originate from N. inopinata.
Nxr Chaperone Constructs Construct Affinity Tags Conditions of Expressed Protein BL21, YT; 0.2, 0.5 µM IPTG; 37°/18°C on; anaerobic Chap_p3NH N-6H BL21pLysS; ZY; 37°/18°C on; aerobic R2pLysS; YT, ZY; 0.2, 0.5 µM IPTG; 37°/18°C on; aerobic and anaerobic R2pLysS; YT; 0.2, 0.5 µM IPTG; 37°/18°C on; Chap_pETM33 N-6H, N-GST aerobic native native DNA BL21; YT, ZY; 0.2, 0.5µM IPTG; 37°/18°C on; aerobic Chap_pETM44 N-6H, N-MBP R2pLysS; YT, ZY; 0.2, 0.5 µM IPTG; 37°/18°C on; aerobic and anaerobic Chap_pCOLADuet-1 - no expression Chap_pCOLADuet-1 C-6H no expression BL21, YT; 0.2, 0.5 µM IPTG, 37°/18°C ITPG, anaerobic BL21; YT, ZY; 0.2, 0.5µM IPTG; 37°/18°C on; Chap_p3NH N-6H aerobic BL21pLysS; ZY; 37°/18°C on; aerobic R2pLysS; YT, ZY; 0.2, 0.5 µM IPTG; 37°/18°C
codon codon optimized on; aerobic and anaerobic Chap_pETM22 N-trx, N-6H Chap_pETM33 N-6H, N-GST Chap_pETM44 N-6H, N-MBP
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MCO-Constructs Soluble protein of MCO_p3NOS_CH_ccdB from Ca. N. inopinata and N. moscoviensis were both weakly expressed in BL21, LB, 0.5 µM IPTG and 37°/18°C overnight. The same was observed during large scale expression (see chapter 3.3 for details).
Because of the low expression yield of the construct MCO_p3NOS_CH_ccdB, expression conditions of the codon-optimized construct MCO_p3NH were also tested in small-scale. The cell lysate and the periplasmic fraction were analyzed separately. In the periplasmic fraction no expression was detected. The analyses of the cell lysate, however, showed consistent expression upon ITPG induction (fig. 6). However, the bands on the SDS-PAGE run at a size of 120 kDa and therefore lower than the expected 175 kDa of MCO. The sample was then sent for mass spectrometry, which confirmed that the band was not MCO but beta-galactosidase (116.4 kDa, UniProtKB – P00722) from the lac operon.
Tab. 14: Overview of small-scale expressed MCO-constructs. All constructs originate from N. inopinata except constructs marked with an asterix (*), which originate from N. moscoviensis.
MCO Constructs Construct Affinity Tags Conditions of Expressed Protein
MCO_p3NOS_CH_ccdB N-strep, C-6H BL21, LB, 0.5 µM IPTG, 37°/18°C on
native MCO_p3NOS_CH_ccdB* N-strep, C-6H BL21, LB, 0.5 µM IPTG, 37°/18°C on MCO_p3NH N-6H no periplasmic expression codon
optimized optimized MCO_p3NH N-6H no expression in cell lysate
Fig. 6: SDS-PAGE of the cell lysate of MCO_p3NH. The expected size of MCO is 175 kDa. Mass spectrometry confirmed that the highlighted bands are overexpressed beta galactosidase from lac operon.
21
CynS-Constructs The initial screening with cynS_p3NOS-CH_ccdB showed no expression. The same approach as with the chaperone constructs (recloning in vectors with solubility tags), however, lead to overexpression of cyanase (fig.7).
Tab. 15: Overview of small-scale expressed cynS- constructs. All constructs originate from N. gargensis.
Cyanase Constructs Construct Affinity Tags Conditions of Expressed Protein cynS_p3NOS_CH_ccdB N-strep no expression cynS_p3NH N-6H R2pLysS; YT, ZY; 0.2, 0.5µM IPTG; 37/18°C on BL21; YT, ZY; 0.2, 0.5µM IPTG; 37/18°C on cynS_pETM22 N-trx, N-6H BL21pLysS; YT, ZY; 0.2, 0.5µM IPTG; 37/18°C on R2pLysS; YT, ZY; 0.2, 0.5µM IPTG; 37/18°C on
Fig. 7: SDS-PAGE of the constructs cynS_p3NH and cynS_pETM22 transformed in BL21. The thioredoxin (trx) in the pETM22 vector increased expression of soluble cynS, while the N-terminal His-tag (cynS_p3NH) couldn’t improve expression.
3.3 Large scale expression and purification Nxr-Constructs Based on the small-scale expression analyses, NxrB_p3NOS_CH_ccdB from Ca. N. inopinata and N. moscoviensis were expressed (BL21, LB, 0.5 µM IPTG, iron sulfur mix, 37°/18°C on). The protein eluted as a single peak from the Strep-tag column, but in the SDS-PAGE two bands were visible (fig.8). Mass-spectrometry revealed that the lower band is indeed NxrB but it was co-purified with the 60 kDa chaperone GroEL (UniProtKB-P0A6F5). GroEL is a double-ring cylinder, that binds unfolded substrates. With its cofactor GroES it forms a chamber around the bound protein. This enables unimpaired folding of the protein without aggregation (45). 22
From this result we concluded that the protein eluted unfolded. Therefore, in the next step, NxrA with the N-terminal Tat-signal peptide, will be co-expressed with NxrB to help expression and protein stability. Additionally, a chaperone for insertion of the molybdenum cofactor will be co-expressed.
Fig. 8: SDS-PAGE of NxrB large-scale expression and purification (L = lysat; P = pellet; S = supernatant; FT = flow-through; E = elution). NxrB elutes together with the 60 kDa chaperone GroEL.
MCO-Constructs MCO_p3NOS_CH_ccdB from both Ca. N. inopinata and N. moscoviensis were transformed and expressed (BL21, LB, 0.5 µM IPTG, 20 µM CuCl2, 37°/18°C on). The elution peaks after Strep- Trap were very small and in the SDS-PAGE no protein could be detected.
CynS-Constructs Based on the results of the small-scale expression, the plasmid cynS_pETM22 was transformed in R2pLysS and expressed in ZY at 37°C / 18°C overnight. The first purification steps consisted of a HIS-Trap, combined with 3C protease cleave over night and a subsequent Rev-HIS-Trap (fig.9). Due to technical issues the His-Trap run had to be executed as a manual run and the chromatogram couldn’t be saved digitally. The chromatogram of the Rev-HIS-Trap shows that the protein with the cleaved off trx- and His-tag does not bind to the column (blue). CynS eluted in two main peaks from the size exclusion column (fig. 10). SDS-PAGE showed that in both peaks there is a band of 18.4 kDa, which is the size of the desired protein. SEC-MALS was performed to check the molecular mass of the proteins in the two peaks.
23
Fig. 9: Reverse HisTrap elution profile of cynS (left) and SDS-PAGE (right). (L: lysis; P: pellet; S: supernatant; FT: flow-through, E: elution) After reverse HisTrap the cleaved off trx-tag and a part of non-cleaved cynS eluted in the second peak (grey), whereas the successfully cleaved cynS is in the flow-through fraction (blue).
Fig. 10: SEC-Elution profile of cynS (left) and SDS-PAGE of the pooled fractions (right). The two main peaks (green and orange) contain the cynS protein (18.4 kDa).
3.4 SEC-MALS The peak from the first pool (green) eluted in two peaks, where the first has an additional shoulder. Proteins in the second peak (orange) have an average molecular size of 148.1 kDa, which suggests cynS forms an octamer. The first peak displays molecular weight of 423.7 and 293.9, corresponding to higher oligomeric (16mer and 24mer, i.e. dimers and trimes of
24 octamers) states (fig. 11). The fraction of the second pool eluted in one single peak with corresponding to an octameric state.
Fig. 11: SEC-MALS elution profile of peak 1 and peak 2 from SEC-run. The main peak (orange) is a monodisperse sample. Based on the size, it can be concluded that the protein is an octamer. In the second peak (green) contains different species. A part has an octameric state, while the other two species are a multiple of eight (16mer and 24mer).
3.5 CynS activity assay The Michaelis Menten constant Km is equal to the substrate concentration at which the maximum rate (vmax) is half-maximum (46). Km and vmax are calculated using the double reciprocal plot (Lineweaver Burk plot). In the plot the negative inverse of Km (-1/Km) is represented at the x-intercept and the inverse of vmax is shown by the y-intercept (46). For representing the data in a double reciprocal plot, inverse of the velocity is plotted over the inverse of substrate concentration. The velocity corresponds to the part of the curve where the slope is linear (fig. 12). From the double reciprocal plot with varying substrate concentration (50, 60, 75, 90, 100, 125 µM KNCO) and a constant enzyme concentration of 500 nM cyanase, a km value of 63.15 µM and vmax of 2.14 µM/s could be calculated (fig. 13). Both substrates (cyanate and bicarbonate) act as inhibitors of the enzyme. This inhibitory effect could be observed already at a cyanate concentration higher than 125 µM.
25
A
0,14
0,12
0,1
0,08 405
OD 0,06
0,04
0,02
0 0 5 10 15 20 25 30 35 t [min]
B v = 0.0196 mol / min
0,045 y = 0,0196x - 0,0088 0,04 0,035 0,03 0,025 405
OD 0,02 0,015 0,01 0,005 0 0 0,5 1 1,5 2 2,5 3 t [min]
Fig. 12: (A) Representative illustration of a time series measurement. The enzyme concentration was 500 nM and the substrate concentration 90 µM. (B) Plot of the curve with a linear slope. The slope of the equation corresponds with the velocity, which is used for the double reciprocal plot.
26
1,2 y = 29,535x + 0,4677 1
0,8
0,6 1/v 0,4
0,2
0 -0,02 -0,01 0 0,01 0,02 0,03 -0,2 1/S
Fig. 13: Double-reciprocal plot of cyanase activity at room temperature. Substrate concentration S is given in µM and the velocity v in µM*s-1.
Azide (NaN3), like different other monovalent anions act as inhibitor of cyanase (34). To test the inhibition effect, the reaction was run for 10 min and then stopped with the addition of the Nessler reagent. The concentration of the cyanate was constant at 100 µM, while the concentration of the inhibitor varied. Fig. 14 clearly shows, that with increased concentration of inhibitor the reaction slowed down significantly. At a molar ratio of 1:1 of substrate and inhibitor, the activity drops at 25% compared to the non-inhibited kinetics. At a ratio of 1:5 the activity even drops at 70%.
0,16
0,14
0,12
0,1
405 0,08 OD 0,06
0,04
0,02
0 0 100 200 300 400 500 600
cinhibitor [µM]
Fig. 14: Representation of inhibitory effect of NaN3 on cyanase.
27
To assess the type of inhibition, measurements with a fixed inhibitor concentration were done (fig. 15). The two lines intersect at the y-axis, which means that azide is a competitive inhibitor and that vmax is unchanged. Due to technical difficulties the measurement could not be repeated with different inhibitor concentrations. Therefore, these results need to be as preliminary until further experiments are completed.
no inhibitor inhibitor (50 µM NaN3)
2
1,5
1 1/v 0,5
0 -0,0200 -0,0150 -0,0100 -0,0050 0,0000 0,0050 0,0100 0,0150 0,0200 0,0250
-0,5 1/S
Fig. 15: Double reciprocal plot showing competitive inhibition of cyanase by azide.
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4 Discussion and outlook 4.1 Cloning Since using expression vectors with solubility tags, especially with trx or MBP (petM22 and petM44), helped to increase overexpression of the desired proteins, this approach will be used for the other NxrAB and the MCO constructs (tab. 16-17).
Tab. 16: Planned to be cloned Nxr-constructs
Nxr Constructs GOI Vector Affinity Tags Resistance Origin Description NxrAB p1 C-6H (subunit B) Ampicillin N. inopinata codon optimized N-strep, C-6H NxrAB p1_NOS (subunit B) Ampicillin N. inopinata codon optimized NxrA p2 - Tetracyclin N. inopinata codon optimized NxrA p2-NH N-6H Tetracyclin N. inopinata codon optimized
Tab. 17: Planned to be cloned MCO-constructs
MCO Constructs GOI Vector AffinityTtags Resistance Origin Description MCO pETM22 N-trx, N-6H Kanamycin N. inopinata codon optimized MCO pETM33 N-6H, N-GST Kanamycin N. inopinata codon optimized MCO pETM44 N-6H, N-MBP Kanamycin N. inopinata codon optimized
4.2 Expression After the cloning of the constructs described above, overexpression will be tested. Since solubility tags helped expression of the chaperone as well as the cyanase constructs, I am optimistic that this will also help with the Nxr and MCO constructs.
4.3 CynS activity assay The data presented is derived from measurements at room temperature and pH 7.7. It is known that pH and temperature can have a massive impact on enzyme activity. In the course of the experiments, however, it turned out that the assay used, in which the conversion of cyanate to ammonium is detected by the Nessler reagent, was not sensitive enough. At higher temperatures (35°C) the inhibiting effect of substrate on enzyme could already be detected at a concentration of 90 µM and not only from a concentration of 125 µM. On the other hand, the substrate concentration cannot be lower than 50 µM, otherwise there would be no difference between the background, which comes from the absorption of the Nessler reagent itself. This results in a very narrow measurement range and therefore, this data can only be interpreted as a preliminary result until further experiments with a more sensitive assay have been done.
29
The most sensitive method to measure ammonia concentration is fluorometric method using the active ingredient OPA (ortho-phthaldialdehyde), which reacts with ammonia. The quantification of ammonia depends on the fluorescence of the condensation products with phthalaldehyde (47). This assay will allow ammonia detection at a submicromolar concentration (48).
For the same reason (narrow measurement range), it was impossible to plot the data in a Michaelis Menten kinetic, because data points from the beginning of the kinetic are not obtainable with this assay.
Compared to other known kinetic parameters of cyanase the km value of Methylobacterium thiocyanatum is the only one similar to those of N. gargensis (tab.18). M. thiocyanatum uses thiocyanate as its sole source of nitrogen and sulfur. During the growth on thiocyanate cyanate is formed as an intermediate. The high activity of the cyanase might be a compensation for the inhibiting effect of thiocyanate on cyanase (49). This means, that in contrast to other organisms, M. thiocyanatum does not use cyanase for detoxification of intracellularly produced or extracellularly exposed cyanate, but rather for growth. The same is true for N. gargensis, which uses cyanase in order to utilize cyanate as its sole source of energy, reductant and nitrogen source.
Tab. 18: km-values of different cyanases.
Species Taxonomic Classification Km [mM] Reference Methylobacterium thiocyanatum Proteobacteria 0.07 (49) Escherichia coli Proteobacteria 0.6 (33) Oryza sativa Plant, Monocotyledoneae 0.63 (38) Nitrososphaera gargensis Archaea, Thaumarchaeota 0.06
The inhibitor plots show that azide is a competitive inhibitor, but the kI could not be determined due to technical difficulties. This experiment needs to be repeated, also preferably with the above mentioned more sensitive assay.
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PART II
5 Utilization of comammox in wastewater treatment The elimination of nitrogen compounds is an essential aspect of biological wastewater treatment in maintaining healthy ecosystem in our surface waters. Conventional methods are mainly based on nitrification and denitrification, whereby ammonia is oxidized via nitrite to nitrate. Each step is catalyzed by distinct chemolithoautotrophic bacteria (ammonia to nitrite: ammonia oxidizing bacteria (AOB) and archaea (AOA); nitrite to nitrate: nitrite oxidizing bacteria (NOB)). Denitrification is the reduction of nitrate to nitrogen gas. Both steps demand special conditions: AOBs and NOBs need oxygen as electron acceptor, therefore nitrification tanks need a be supplied with a lot of additional oxygen. Denitrifying bacteria, on the other hand, require anaerobic or anoxic conditions but they need an external carbon source for growth. Engineers need to take all these differences into account to be able to develop an optimal process, where cleaning performance, the conditions for the microorganisms and the operating costs are in equilibrium (4). The first enhancements in wastewater treatment followed the discovery of anammox bacteria. Partial nitritation (oxidation of ammonia to nitrite) is coupled with the anammox process, where ammonia and nitrite are oxidized together to nitrogen gas. This process is, therefore, a shortcut to the conventional nitrification/denitrification process. Another advantage is that anammox bacteria grow under anaerobic conditions, which leads to a more cost-saving operation (50)-(51). The next step in this development could be marked by the discovery of comammox bacteria. They are able to perform complete nitrification. Although comammox are a recent discovery, research on their utilization in wastewater treatment is already being done and could provide another technological progress in this field (9)-(13). In the following excursus, I will first explain the role of nitrification and denitrification in wastewater treatment in general and then use the Vienna Main Wastewater Treatment Plant (VMWWTP) as an example. Furthermore, I will discuss technological progress in connection with deammonification and anammox. Lastly, I will give an outlook on the potential use of comammox in wastewater treatment plants (WWTP).
5.1 The role of nitrification and denitrification in wastewater treatment Nitrification and denitrification are vital processes for nitrogen removal in WWTPs. During the nitrification, ammonia is oxidized to nitrite and nitrate, which is explained in more detail in chapter 1.1. Both steps are producing energy, but also require a lot of oxygen, which means that wastewater treatment plants need to supply nitrification tanks with a lot of air. Carbon for biosynthesis is obtained by CO2 reduction (4).
A successful nitrification process is important. Otherwise nitrifying bacteria would oxidize ammonia causing a decrease in dissolved oxygen, which in turn would be detrimental to
31 aquatic life. Simultaneously, nitrate promotes the growth of algae, which would contribute even more strongly to eutrophication. Uncontrolled nitrification would also lead to a drop in pH levels, which in turn influence the chemical ammonia/ammonium equilibrium (4).
Denitrification is the biological reduction of nitrate into nitrogen gas and therefore acts as a counterpart to nitrogen fixation. The entire denitrification process consists of various, subsequent reduction steps [5-8] with different intermediates. Each reduction step is catalyzed by a specific reductase enzyme (nitrate [5], nitrite [6], nitric oxide [7] and nitrous oxide reductase [8]).
- + - [5] NO3 + 2 H NO2 + H2O
- + [6] NO2 + 2 H NO + H2O
+ [7] NO + 2 H N2O + H2O
+ [8] N2O + 2 H N2 + H2O
The intermediates, nitric oxide (NO) and nitrous oxide (N2O), are potential greenhouse gases.
N2O is predicted to be the main ozone depleting substance of this century. N2O emission will continuously rise due to heavy use of nitrogen fertilizers (52). NO, on the other hand, contributes to the production of ground-level ozone and acid rain (53).
Under anoxic conditions nitrate and nitrite serve as electron acceptors instead of O2, whereas organic compounds serve as electron donors (4). If electron donors become limited, the intermediate products increase. To avoid incomplete denitrification, additional carbon sources as electron donors are added to the wastewater. Methanol, ethanol or acetate are commonly used (54).
Denitrifiers are usually heterotrophic organisms, but not all of them can perform the complete denitrification process. Some lack the enzymes for one of the four reduction steps. Thus, a consortium of denitrifiers with different nitrogen oxide reduction capabilities are commonly found together (55).
A common problem in WWTP is incomplete denitrification and the resulting accumulation of denitrification intermediates. Accummulated nitrite can have two negative influences on the activated sludge system: Firstly, the nitrite can be reoxidized into nitrate. As described above [2], this reaction needs oxygen and also leads to an increased carbon source consumption during denitrification (56). Secondly, nitrite is a toxic compound and might inhibit the denitrification activity (57). Another issue for WWTP is the emission of the greenhouse gas
N2O. Electron competition between the four reduction steps [5-8], as well as various environmental parameters, such as DO concentration, pH and carbon source availability might also cause intermediate accumulation (58).
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5.2 Wastewater treatment parameters The following parameters are used to characterize the degree of pollution or are important process control parameters.
BOD5: biochemical oxygen demand after five days. The BOD5 stands for the mass of oxygen consumed by microorganisms in a sealed flask during a period of 5 days. It is an indicator for the amount of biologically degradable components. It is indicated in mg O2/l water. (4), (59)
COD: chemical oxygen demand. The COD is defined as the mass of oxygen, which is needed for the complete oxidation of an organic compound. It is therefore an indicator for oxygen- depleting elements in wastewater. It is indicated in mg O2/l water. (4), (59)
TOC and DOC: total organic carbon and dissolved organic carbon. TOC stands for the entire amount carbon found in organic compounds. DOC is defined as fraction of organic carbon that passes through a 0.45 µm pore size membrane. Both parameters are indicators of organic pollution of wastewater. (59), (60)
DO: dissolved oxygen. DO is defined as relative measure of oxygen dissolved in wastewater, which is needed by bacteria in activated sludge. (61)
Sludge age / SRT: sludge age / sludge or solid retention time (sometimes solid residence time). Sludge age determines the efficiency of substrate removal in wastewater, whereby the critical sludge age must be exceeded to avoid that bacteria will be washed out. (4)
TN: total nitrogen. TN is the sum of organically bound nitrogen and ammonia-nitrogen
(NH4-N), nitrite-nitrogen (NO3-N) and nitrate-nitrogen (NO2-N). (fig. 16)
NH4-N: Ammonia nitrogen. It is produced during the breakdown of organic nitrogen compounds and is indicated in mg N/l water.
Fig. 16 nitrogen compounds in wastewater. Figure modified after: (4)
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5.3 Wastewater treatment explained on the concept employed on the Vienna Main Wastewater Treatment Plant (VMWWTP) The first Vienna Main Wastewater Treatment Plant was designed in late 1960 and started operation in 1980. It was built as a single-stage activated sludge plant mainly focusing on the removal of carbon. Additionally, around 40 % of nitrogen could be removed by this single- stage plant. In the 1970s, raw sludge and ash disposal were implemented (62), (63). However, the VMWWTP had to be retrofitted in 2005 to meet new Austrian water protection legislation criteria. They mandated a total nitrogen removal rate of 70 % and a maximum NH4-N effluent concentration of 5 mg/l. To improve nitrification and denitrification processes, VMWWTP had to be extended by a second biological cleaning stage (64). The first plant, however, was fully integrated into the extension.
In principle, VMWWTP performs two different purification steps – the mechanical and the biological treatment.
The first stage is mechanical cleaning. The main focus is to reduce the number of solid parts which would increase the wear and tear of the equipment. At first, the wastewater passes through two 12 m long and 5 m wide channels of a gravel trap. Solid parts sink into a recess in the bottom of the floor, where they get removed and incinerated. On average, 15 t are removed each week (65). Up to six screw pumps pump wastewater up to plant level, which is 5.5 m higher than the sewer level. At this level, wastewater can flow through the plant only by gravity and no further pumps are needed. During dry weather 680000 m3 and at peak periods up to 1,500,000 m3 of wastewater are transported by the screw pumps (66). The second installation of the mechanical cleaning process is a screening chamber. Coarse (8 mm) and fine (3 mm) screens remove plastic bags, sanitary items, etc. Extracted items get dewatered and incinerated. This step removes about 10 to 15 t per day (67). In a sand trap, the velocity of the wastewater gets reduced to 10 cm/s. It consists of six tanks, which are 3.6 m deep, 4 m wide and 48 m long. This allows fine-grained particles like sand, ashes, etc. to settle down on the bottom of the tank. A scraper then pushes these sediments into a hopper. This step removes approximately 5 t of fine sediments each day (68). In the last step of mechanical cleaning, the wastewater is going through the primary clarifier or primary sedimentation tanks. In four tanks of 70 m length and 33 m width, the flow rate is further reduced to 2 cm/s. This decreased velocity allows even flaky sediments and smaller particles to sink to the bottom. The sediments of this stage are called primary sludge and every day between 80 to 120 t of primary sludge is removed and transported to thickening tanks. After the mechanical cleaning process, 30 % of impurities are removed. In the following biological cleaning stages, remaining dissolved impurities are further removed (69).
The biological cleaning stage begins with treatment in the first aeration tanks. The main purpose of this stage is the removal of carbon, nitrogen and phosphorus compounds by microorganisms (activated sludge). This happens in four tanks (73.5 m long, 19 m wide and 6.8 m deep) that can contain up to 51,000 m3 of wastewater. The dissolved impurities from the primary sedimentation serve as nutrients for different microorganisms. To metabolize
34 these compounds a lot of oxygen is needed. Central aerators immersed in the tanks blow in a substantial amount of air. This creates a turbulent motion, which keeps the wastewater and the activated sludge constantly mixed. From the intermediate sedimentation tanks return sludge is fed back to the aeration tanks, which ensures constant and sufficient supply of biomass. But this also results in 100 t excess sludge each day. A precipitant agent is added to convert dissolved phosphorus into insoluble compounds. This is then removed together with the excess sludge. After the wastewater has passed the aeration tanks 80 % of the carbon and 35 % of the nitrogen content has been removed (70).
After the aeration tanks, the wastewater passes through the intermediate sedimentation stage. The main purpose of this stage is the separation of activated sludge and wastewater. The 24 tanks are 73 m long, 11 m wide and 6.45 m deep. The flow rate is reduced to 1 cm/s, which allows sludge to settle down at the bottom. The main portion is pumped back into the aeration tanks, where microorganisms are exposed to raw wastewater. Excess sludge is withdrawn and transported to sludge thickening tanks (71).
The second biological stage is done in 15 tanks with a length of 79 m, a width of 33 m and a depth of 5.5 m. The main focus is the removal of nitrogen. By dividing the tanks into different zones, areas with either high or low oxygen content can be established. This division is required by different microorganisms to perform nitrification (aerobic conditions) and denitrification (anoxic conditions). Each tank consists of three cascades and one degassing zone: The first cascade is not aerated, instead, two vertical mixers prevent the sludge from setting down. The second and third cascades vary in oxygen amount, which depends on the level of pollution and wastewater temperature. To adjust air supply, 48000 membrane diffusers are installed at the bottom of the tanks. Together with submersing mixers, they assure that wastewater and active sludge stay a homogenous unit. At the end of the denitrification process stands gaseous nitrogen. In the degassing zone, large air bubbles remove the remaining gaseous nitrogen from the wastewater (72).
The two-stage biological cleaning allows for different operational modes: Hybrid®-, Bypass- and conventional two-stage operation which is the one described above. In the Hybrid®-mode, part of the activated sludge from stage 1 (first aeration tanks) is exchanged with sludge from stage 2 (second aeration tanks) (73). This is possible due to two additional sludge circulation lines, where exchange sludge flow never exceeds a ratio of 5 %. For the denitrification process in the second stage, anoxic conditions and an additional carbon source are essential. However, the main portion of carbonaceous compounds are removed in the first stage and this leads to a problem for denitrifying bacteria. During the removal of carbon in the first stage, approximately 80 % of the total COD is incorporated in the activated sludge. The sludge from this first stage is, therefore, an excellent carbon source. This means that the carbon source for denitrification does not come from the pollution in the wastewater itself (BOD5) but from the exchanged activated sludge. Return sludge containing nitrifying bacteria is pumped into the first stage, reducing the nitrogen load already in this stage (fig. 17). The key factor for this
35 operation mode is the control of biomass transfer. The appropriate sludge age needs to be reached for full nitrification (64).
Fig. 17: Flow scheme of the Hybrid®-mode. Figure modified after: (73)
The second operation mode is the Bypass-mode (fig. 18). As the name suggests, part of the incoming wastewater is bypassing the first stage and flowing directly into the second stage. The bypass-flow ranges from 10 % to 40 %. This means that raw, carbon-rich wastewater provides a substrate for denitrifying bacteria in the second stage. Bypassing wastewater also results in different loads at different stages. If the bypassing flow is increased, the load on the second stage will increase, while decreasing in the first stage. If the incoming flow is too high, the sludge age in the second stage will decrease. This, in turn, results in an insufficient sludge age for the growth of nitrifying bacteria. Effluent from the final clarifier as well as excess sludge from the second stage is sent back to the first stage. This means that denitrifying bacteria from the sludge and nitrate-rich water come back into the first stage. Together with fresh incoming wastewater, which provides enough carbonaceous compounds for denitrifying bacteria, optimal conditions for denitrification are created.
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Fig. 18: Scheme of Hybrid®- and Bypass-mode. AT = aeration tank, ES = excess sludge, RF = external recirculation, RS = return sludge, SC1 and SC2 = activated sludge exchange during Hybrid®-mode, SST = secondary sedimentation tank. Figure modified after: (63)
It has been shown that the conventional mode could not reach the nitrogen removal requirements. This is mainly due to the lack of sufficient carbon source for denitrification in the second stage. To avoid this problem, Hybrid®- and Bypass-mode return/exchange sludge of the two stages and return nitrate-rich water to the first stage. Bypass-mode is better for nitrogen removal, which is due to temperature effects and nitrate-rich water returned to the first stage. The disadvantage of the Bypass®-mode is the decreased stability of nitrification. This is caused by two things: Firstly, directly fed wastewater in the second stage can inhibit nitrification bacteria. Secondly, the increased influent on the second stage can decrease sludge age (64).
At the end of the cleaning process, the wastewater stays for 4h in the final clarification tanks. The activated sludge sinks to the bottom and is removed by scrapers. The purified water is regularly monitored before it leaves the wastewater plant into the Danube Canal (74).
In 2020 the VMWWTP will implement the so-called EOS project. EOS stands for energy optimization of sludge treatment and this will allow the plant to become entirely energy self- sufficient. Around 1 % of Vienna's overall electricity demand is consumed by the VMWWTP. Around three-quarters of this energy is used by microorganisms to remove pollutants from the wastewater.
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Fig. 19: 2-stage activated sludge plant with primary sedimentation. AT: aeration tank, SST: secondary sedimentation tank. Reject water from the dewatering stage is purified by nitritation and is then directed into the main process circle of the plant. Figure modified after: (63) Activated sludge contains a lot of energy. Coming from the primary sedimentation tank and the first aeration tank, the sludge has high water content. It needs to get partially dewatered before entering the digesters (fig. 19). The crucial part is to find the perfect sludge consistency for this process. On the one hand, water has to be extracted from the sludge, so that the heating to 38°C in the digester does not consume too much energy. On the other hand, the sludge must not be too thick, otherwise, it can’t be pumped through the system. When the pre-thickened sludge in the digester is constantly kept in motion and heated to 38°C, a lack of oxygen allows anaerobic bacteria to break down organic compounds. During the digestion (stabilization) phase, which takes 25 days, biogas is produced in the digesters. This gas consists 3 of two-thirds of methane CH4 and is stored in gas tanks. It is estimated that 20,000,000 m of methane could be produced by this process. The methane is then combusted in combined heat and power stations by gas engines, which drive generators to produce energy. The produced energy does not only cover the energy demand of the plant itself, but unconsumed electricity will be fed into the public energy grid. The stabilized sludge is pumped to a sludge storage tank and subsequentially to the dewatering unit. The water from the dewatering process is called reject water and contains approximately 25 % of the total nitrogen influent load. The VMWWTP decided to purify nitrogenous compounds in reject water by partial nitritation and denitritation in the first stage of the plant. Another possible way to purify this water is deammonification, which will be explained in the following chapter (chapter 5.4-5.5). The main reasons for the decisions were (63):
Short start-up phase without seed sludge Nitritation is a well-known and easy process. 50% conversion without pH control and up to 80% with lime addition for pH control can be reached. Denitritation-process saves aeration energy for carbon removal. The oxygenation efficiency in the reject water is significantly higher than in the first aeration tanks. 38
The first aeration tanks contain enough carbon for full denitritation.
The treatment of this reject water is the main focus of this study, since it does not have to be implemented in the main wastewater treatment plant and could be operated separately. In the following chapters, I want to discuss different methods for nitrogen removal from ammonia-rich reject water (tab. 19), as well as answer the question whether comammox bacteria could be used for this task.
Tab 19: Overview of WWTP parameters.
Conventional Deammonification VMWWTP ANAMMOX® DEMON® DeAmmon® ANITATMMox solids retention time (SRT) / sludge age 1 -6 days 35 days 30 days x x temperature (T) 8-22°C 30-40°C 25-30°C 27-30°C dissolved oxygen (DO) 1.5-2.5 mg/l 0.3 mg/l max. 3.0 mg/l 0.5-1.5 mg/l pH 6-8 7-8 7-7.1 7.3-7.7 6.7-7.5
NH4-N influent 60 mg/l 500-1000 mg/l 500-1000 mg/l 1000 mg/l
NH4-N effluent 5 mg/l 25-50 mg/l 50-200 mg/l 100 mg/l (75), (76), (77), references (63) (78), (79, 80) (81), (79), (80) (79) (79), (80)
5.4 Anammox and Deammonification Anammox stands for anaerobic ammonia oxidation. In this process the anaerobic oxidation of ammonia into nitrite is coupled with the reduction of nitrite to nitrogen gas [9]. It has also been shown that nitrogen gas is produced from equimolar amounts of ammonia and nitrite (82). Even though the anammox process is energetically more favorable than aerobic ammonia oxidation, microorganisms capable of performing this process remained unknown for a long time (83).
+ - [9] NH4 + NO2 N2 + 2 H2O
When they had finally been discovered, it became clear, that some key morphological and physiological characteristics must differentiate these bacteria from known AOB or NOB: Aerobic oxidation of ammonia into nitrite is performed in two steps, where the intermediate hydroxylamine is further oxidized into nitrite [1-2]. During the anammox process, however, ammonia reacts with hydroxylamine to produce hydrazine (fig. 20) [10]. Hydrazine is an energy-rich, but potentially toxic compound (84). Another unique feature of anammox bacteria is their membrane-bound compartment. This intracellular compartment is called anammoxosome and is the place where the anammox catabolism takes place. The anammoxosome not only contains large quantities of a hydroxylamine oxidoreductase-like enzyme (85) but also a true hydrazine-oxidizing enzyme (86). Furthermore, anammox bacteria
39 contain large amounts of cytochrome c type proteins, which makes them look red. 16S rRNA analysis has confirmed, that anammox bacteria belong to the order of Planctomycetales; so far nine different species within five genera have been discovered (87). In contrast to nitrifying bacteria, anammox form flocs or granules, which makes them easier to separate from other microorganisms.
+ + [10] NH2OH + NH4 N2H4 + H2O + H
Fig. 20: Possible reaction mechanism of anammox bacteria, where ammonia and hydroxylamine react together to produce hydrazine. Figure modified after: (85).
Anammox play a key role in the so-called deammonification, which is a combination of the biological oxidation of ammonia to nitrite (nitritation) and the above described anammox (50) (88) process (anaerobic ammonia oxidation). The first part is also called partial nitritation, since only half of the total ammonia is converted into nitrite. As described above, during the anammox process, both (ammonia and nitrite) are anaerobically oxidized to nitrogen gas. (fig. 21) (79).
- Fig. 21: Schematic illustration of deammonification. NH3 is partly converted to nitrite (NO2 ) by aerobic ammonium oxidizers. The produced nitrite is used as electron acceptor by anammox bacteria, which anaerobically oxidize ammonia to nitrogen gas
(N2). Figure modified after: (79).
Deammonification is often used to treat reject water from dewatering systems (89). As mentioned before, this water is rich in ammonia as it carries up to 20-25 % of the total ammonia-load. The process is used for the following reasons (79): 40
1. Since anammox bacteria oxidize ammonia under anaerobic conditions, less oxygen has to be supplied, which results in a 55-60 % decrease of aeration energy. 2. In conventional WWTP nitrogen gas is produced via denitrification, which is not the case during deammonification. Therefore, no additional carbon source has to be provided throughout the process. 3. Compared to the conventional nitrification/denitrification process deammonification
is a net consumer of CO2. 4. Sludge production is reduced. 5. The demand for alkalinity is reduced by about 45 %.
5.5 Deammonification process technologies Deammonification is a powerful and cost-saving alternative to conventional nitrification and denitrification in WWTP. There are three main technologies used by the industry; they set themselves apart by the way they accumulate sufficiently slow-growing anammox bacteria, the way they retain them in the system as well as by the number of stages, process configurations and control strategies. The following three technologies are mainly used:
5.5.1 ANAMMOX® Granulated Sludge Reactor As mentioned before, anammox bacteria grow in granules, which makes it easier to separate them from other microorganisms. The granular sludge reactor design takes advantage of the fast setting rates of anammox granules by using a high rate clarifier. Therefore, anammox granules are retained, which results in an increased sludge age, while competing bacteria can be washed out. One of the advantages of this design is that the volumetric loading rate is higher than in other anammox reactor types. The conversion rate depends on the surface area of the biomass, its thickness, the nitrite level and the penetration depth in the biofilm. In this case, granules have a very high specific surface area, which can be around 3,000 m²/m³. The surface area is directly proportional to the conversion rate. This technology can be arranged in a single-step or two-step set-up (90).
The two-step ANAMMOX® process combines the SHARON (78), (91) (single reactor high activity ammonia removal over nitrite) with the anammox process (fig. 22, tab.19). The SHARON process is designed to treat ammonia-rich reject waters at a high temperature between 30-40°C and at a pH between 7-8. At these elevated temperatures (normal WWTP temperatures would be 5-20°C) ammonia oxidizers grow faster than nitrite oxidizers. Together with the missing sludge retention, ammonia oxidizers grow faster and can, therefore, be kept in the system by controlling the hydraulic retention time. Nitritation decreases the pH (per + + mol NH4 , two moles H are produced). CO2-stripping neutralizes about 50% of the produced acid (78). The SHARON process is specifically attractive for plants with a limited aeration capacity, limited denitrification capacity or a limited aerobic sludge age (91). In the second tank anammox bacteria in granules are kept by upflow clarification. The conversion rate is
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approximately 4.8 . The advantage of separating the SHARON and the anammox process ∗ is the better control of the entire process. On the other hand, this design demands a lot of space due to the larger reactor volumes.
Fig. 22: Two-step ANAMMOX ® process: Nitritation happens in the first, aerated tank (SHARON), which is separated from the tank with anammox bacteria. In the latter, ammonia and hydroxylamine is oxidized together to N2 under anoxic conditions. A high rate clarifier in the second tank captures the anammox flocs and retains them. Figure modified after: (79)
Fig. 23: Single-step ANAMMOX ® process: Partial nitritation and anammox processes taking place in one tank. Stable operation is guaranteed by keeping a balance between aerobic phases for AOBs and anoxic phases for anammox by controlling aeration time. Figure modified after: (79)
In the single-step ANAMMOX® process oxidation of ammonia to nitrite as well as anammox happen in a single tank (fig. 23, tab.19). One challenge of this set-up is to achieve a sufficient sludge age for anammox bacteria to grow, but also to be able to wash out other competing microorganisms. Therefore, process parameters such as pH, DO (dissolved oxygen) and ORP (oxidation-reduction potential) have to be constantly controlled. The advantage of this design is undoubtedly the compactness of the entire set-up. At the same time, however, it is much more complicated to control and to run a stable process in just one tank. The key to success is to maintain a balance of aeration for nitritation as well as anoxic conditions for anammox bacteria to produce nitrogen gas (92).
5.5.2 DEMON® Sequencing batch reactor In principle, a sequencing batch reactor consists of one or more tanks with five essential periods. These are: fill (incoming raw wastewater), react (time to complete reaction), settle (time to separate sludge from effluent), draw (discharge of effluent) and idle (time between
42 discharge of effluent and refill with fresh raw wastewater). As the name suggests, a sequencing batch reactor operates in batch mode, as opposed to a continuous mode (93).
Another technology that takes advantage of the anaerobic oxidation of nitrite to nitrogen gas is the DEMON® reactor (fig. 24, tab.19). DEMON® is an acronym and stands for DE- amMONnification. Like in the single-step ANAMMOX process described above, both steps (aerobic oxidation of ammonia to nitrite and anaerobic oxidation of ammonia and nitrite to nitrogen gas) happen in one tank. To make this system work, several challenges have to be overcome: Firstly, aerobic ammonia oxidizers grow very slowly and might be outcompeted by other bacteria. Secondly, the microorganisms needed for these two steps require different conditions, and what benefits one group might inhibit the other. Nitrite is necessary for anaerobic oxidation by anammox bacteria, but is toxic already in low concentrations. Oxygen is needed by the ammonia oxidizers, but inhibits anammox bacteria. To guarantee a stable process, it is essential to control certain parameters: The pH is regulated by the aeration cycle. During the nitritation phase, the tank is supplied with oxygen, which leads to a decrease in pH. Whereas in the anammox phase, the tank is not aerated and the pH starts rising again. This means that the aeration period starts, when the pH reaches its upper level and stops when it reaches the lower level. The length of the aeration period also regulates the growth of nitrite oxidizing bacteria (NOB). A shortening of the aeration cycle, followed by an anoxic mixing period inhibits NOB activity. The concentration of dissolved oxygen must be kept at a low level (approximately 0.3 mg/l) to prevent rapid nitrite production and to repress NOBs. If a stable process is implemented it leads to an average energy saving of around 60 %. Another strong argument for the DEMON® process is its positive CO2 balance (94).
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Fig. 24: DEMON ® Sequencing batch reactor. (1) Aeration phase; (2) fill/react phase; (3) settling phase; (4) discharge phase. Figure modified after: https://www.rmwea.org/docs/Deammonification_PWO_Seminar_20190207.pdf
5.5.3 Moving Bed Biofilm reactors (MBBR) MBBRs are the third main application used for wastewater treatment in connection with anammox bacteria. The key of this technology lies in the thousands of submersed carriers, on which microorganisms can build biofilms. These biofilms consist of two zones: aerobic bacteria form the outer layer, which means they are exposed to DO in their surroundings, while anaerobic bacteria are protected in the inner layer (fig. 25). Carriers have to be kept in constant motion to guarantee the availability of substrate and oxygen for the microorganisms in the biofilm. They are typically made from polyethylene and designed to maximize the surface area on each individual piece. Screens between different stages retain the carriers but allow the treated effluent to flow from one tank to another tank (fig.26) (95).
Fig. 25: Two zones of biolayer on a carrier. Aerobic bacteria form the outer and anaerobic anammox bacteria the inner layer Figure modified after: (79). 44
The benefits of MBBRs can be summarized as followed (96):
Tank volume is significantly smaller than conventional WWTP at a performance comparable to active sludge processes. Retention of biomass is independent of clarifiers. MBBRs are operated as continuous-flow processes, therefore, the biomass thickness on the carriers is controlled by air flow or mechanical mixing energy. MBBRs are suitable for retrofit installations. Liquid-solids separation can be achieved with a number of different methods (e.g. sedimentation tanks, membrane filtration etc.)
Fig. 26: Schematic diagram of a moving bed biofilm reactor with carriers in the aerobic and anoxic zones. Figure modified after: (97) Three main set-ups of MBBRs are used in WWTP: DeAmmon®, ANITATMMox and Terra-N®. DeAmmon® consists of a single or dual reactor with three stages per reactor (tab. 19). It is operated with intermittent aeration for aerobic oxidation of ammonia (20-50 min), followed by an anoxic phase for anerobic oxidation (10-20 min). During the aerobic stage, the dissolved oxygen concentration is kept at 3.0 mg/l. The DO concentration must not be higher, otherwise nitrite accumulations or growth of competing nitrite oxidizers cannot be avoided. During the anoxic stage mechanical agitators keep the wastewater and the carriers well mixed to assure the availability of substrate to the microorganisms. Even though pH is not controlled, conductivity is measured to monitor the performance and if necessary, to adjust aeration time. Operation temperature is between 25-30°C, which makes this process suitable for industrial wastewater treatment at higher temperatures. Separating screens prevent the carriers from being washed out (98) (99).
ANITATMMox is a single stage MBBR system and similar to the DeAmmon® process, except that ANITATMMox is operated in constant aeration mode (tab.19). The DO is set between 0.5-1.5 mg/l and the average operating temperature lies between 27° and 30°C. An online measurement of the ammonia and nitrate concentration regulates the DO. Another difference lies in the used carriers that are used: ANITATMMox uses specific Biofilm Chips, which have an increased surface area. ANITA®Mox saves even more energy, because the DO level is 45 constantly lower than in the aeration phase of the DeAmmon® process and because there is no need for mixers due to the constant aeration, which keeps the carriers in constant movement (100).
The Terra-N® technology is designed as single-stage SBR (sequencing batch reactor) and uses bentonite instead of polyethylene carriers. The particles vary in size from 20 to 45 µm. With the addition of bentonite, the solids settling rate increases as well as the compactness of settled solids. On the one hand, this leads to some losses of bentonite in the effluent. On the other hand, it is sufficient enough to guarantee a controlled reactor solids concentration. Tanks are intermittently aerated; aeration time is adjusted according to measured ammonium load and reactor performance. During the anoxic stage, mechanical mixers keep solids and wastewater homogenized (92).
5.6 Comammox and WWTP As shown before, the discovery of anammox bacteria has revolutionized our understanding of the nitrogen cycle. Furthermore, it has led to the development of new technologies, which have had far-reaching consequences in the wastewater industry. The recent identification of comammox Nitrospira has opened up another chapter that may once more change modern wastewater treatment. Comammox bacteria are the first known organisms capable of complete ammonia oxidation. This falsified the century-long paradigm that nitrification was a two-step process, performed by coordinated activity of distinct groups of bacteria (ammonia- oxidizing bacteria and archaea (AOB and AOA) and nitrite-oxidizing bacteria (NOB)) (9) (13). Before a possible application of comammox in WWTP can be discussed, it is important to have a closer look at this new type of bacteria and to answer the questions of what sets them apart from other microorganisms and of how they could be used in engineered systems.
A study from 2016 compares nine WWTP (conventional activated sludge and deammonification systems). They analyze 16S rRNA gene copies of comammox Nitrospira and conclude that Nitrospira-like comammox were not ubiquitous in wastewater treatment and that their relative occurrence compared to other ammonium-oxidizing bacteria was low (101). This statement, however, might be incorrect, as “comammox Nitrospira do not form a monophyletic group in 16S rRNA gene sequence or nxrB-based phylogenetic analyses, but are instead interspersed with strict nitrite-oxidizing Nitrospira” (102). This observation is also in contradiction with the initial report of comammox Nitrospira’s discovery. According to FISH analyses of samples taken for the WWTP VetMed in Vienna, Nitrospira species outnumber AOBs, and comammox represent between 43 and 71 % of the Nitrospira population (9).
All known comammox belong to the genus Nitrospira. One prediction about comammox is that they are adapted to microaerophilic or anaerobic conditions (103). These conditions are very hard to control and hard to achieve with operational technology. That Nitrospira require microaerophilic or anaerobic conditions is based on several observations:
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Possession of a reductive tricarboxylic acid cycle: Comammox, as well as all Nitrospira, encode genes for the reductive tricarboxylic acid (rTCA) cycle (15) with two, highly oxygen-sensitive key enzymes (2-oxoglutarate:ferredoxin oxidoreductase and pyruvate:ferredoxin oxidoreductase) (104). The rTCA cycle is mainly found in anaerobic organisms. Structure of the electron transport chain: The novel cytochrome c oxidase (cytochrome bd-like terminal oxidase) (15) is thought to have a higher oxygen affinity and therefore enables comammox to adapt to low dissolved oxygen concentrations. Response to oxidative stress: Candidatus Nitrospira defluvii, for example, lacks key enzymes (catalases, superoxide dismutases, superoxide reductases) for protection against reactive oxygen species (103), which are found in most aerobic organisms (15).
Comammox’ adaptation to microaerophilic or anaerobic conditions has been confirmed in experiments by Roots et. Al (103), who have found that nitrification was still efficient at low DO concentrations of 0.2 to 1.0 mg/l. Camejo et al. (105) have enriched comammox Nitrospira in a low DO reactor, which had been inoculated with activated sludge. A paper from 2020, however, even reported stable abundance at exceeding 2.0 mg/l DO concentrations. Furthermore, it claims that there is no correlation between DO concentration and comammox presence or absence respectively (106).
According to kinetic and thermodynamic analysis comammox Nitrospira is predicted to have a high growth yield and a low specific growth rate(107)-(12). In fact, their Km value (Km = 0.049 - µM NH3) is 4-2500fold below reported values of AOBs. The affinity for NO2 on the other hand, - is worse compared to other known NOBs (Km(comammox) = 449.2 µM NO2 ; lowest Km(NOB) - = 9-27µM NO2 ) (23). This indicates that they are best suited for an oligotroph lifestyle with a limited substrate influx. Incidentally, comammox have indeed been discovered in these very conditions, namely in biofilms (9), (13).
During heterotrophic denitrification and chemolithoautotrophic aerobic nitrification the trace gases nitrous oxide (N2O) and nitric oxide (NO) are formed. Wastewater treatment contributes up to 3.4% of global N2O emissions. Based on a mathematical model, comammox are predicted to produce less NO and N2O (108). Kits et al. (109) have shown, that Ca. N. inopinata lacks enzymes for denitrification (NO reductase) to N2O and that it has a low N2O yield, which is also independent of oxygen levels. Compared to canonical AOBs, Ca. N. inopinata produces less N2O and it also does not release NO during hypoxia.
To summarize, the following parameters have to be taken into consideration for process design with comammox: (1) low DO level (2) long sludge retention time and (43) limited substrate availability. Now that these prerequisites have been established, the question that remains is: If and how could comammox Nitrospira be utilized in Vienna’s main wastewater treatment plant?
The main wastewater treatment process is a complex system consisting of several coordinated stages (see chapter 5.3). Although the sludge treatment of the EOS extension is integrated
47 into the main process, the treatment of the resulting reject water can be seen as a separate component. Reject water has a very high ammonium load (25% of the total N load) and a common process to treat it is partial nitritation coupled with the anammox process (see + - chapters 5.4, 5.5). Since the affinity of comammox to NH4 is much higher than to NO2 (107), it would theoretically be possible to use commamox instead of canonical AOBs, which are now responsible for the oxidation of ammonia to nitrite. The advantage being, that comammox do not require high concentrations of DO, which would also agree with the conditions needed by anammox bacteria. Therefore, aeration for partial nitritation – as needed in conventional deammonification processes - would not be necessary, leading to cost-savings. As anammox and comammox prefer similar environments, this would also result in a simplified plant design where both processes could be performed in one tank.
Unfortunately, this approach will not work in practice due to the high substrate concentration in the reject water (reported values range from between 300 to 1600 mg NH4-N/l) (103), (63). If comammox were to be used in such a process, the reject water would have to be strongly diluted to lower substrate concentration. One way to achieve this would be to mix in other partial streams of the process that contain less ammonia. This would, however, not lead to cost-savings and simpler design, but rather to the opposite.
Wu et al. combined aerobic partial nitritation, anammox and comammox to treat ammonium rich reject water (500-2000 mg/l) in a SBR (110). In stage 1 the aerobic oxidation of ammonium to nitrite takes place. The tank is aerated for 11 hours while the DO concentration is kept between 2-4 mg/l. In this time 55 % of the nitrogen is removed by AOBs. Stage 2 is the anammox phase and lasts for 6 hours. Aeration is stopped, which leads to a decrease in DO concentration to under 1 mg/l and 18 % of the total nitrogen is removed. The following stage 3 lasts with 27 hours the longest. Total nitrogen is further removed by 27 %. As in phase 2, the tank is not aerated during the comammox phase to maintain the anoxic conditions. The overall nitrogen removal rate of this combined process lies around 98 %. Even though this experiment is at the beginning of this research field, it has to be stressed that the produced results reflect lab conditions and a full-scale implementation of this process has still to be explored. Another disadvantage of the combination of all three processes in one operation mode is process stability. All three stages require different conditions (different substrates, substrate concentration, DO concentration), and a stable equilibrium between them will be essential for implementation in a WWTP.
Even though its use in reject water treatment does not appear to be feasible at present, comammox’ application in the main process should still be considered. Here, they could also be used instead of canonical AOBs for oxidizing ammonium to nitrite under low DO concentrations. As in the separate component approach, this would also lead to lower aeration costs. The annual average of total nitrogen concentration in the main process is approximately 60 mg NH4-N/l (63). Even though this concentration might still be too high for commamox, it would seem, that with the Hybrid- and Bypass-Mode in the VMWWTP, the nitrogen load could be better distributed throughout the system. This would theoretically
48 enable comammox to outcompete canonical AOBs. To achieve this, a much longer SRT seems essential. While the average SRT in the second purification step is now around 6 days, research indicates that 99 days would be needed.
In the concrete example of Vienna’s main wastewater treatment plant, an operational switch to comammox does not appear to make sense in its present setup. Further research into the application of commamox in WWTP will be necessary to plan or adapt plants accordingly in the future. It is already clear now, however, that comammox is an excellent choice for niche applications due to its metabolic diversity and its high degree of specialization.
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6 Abbreviations AMO Ammonia monooxygenase AOA Ammonia-oxidizing archae AOB Ammonia-oxidizing bacteria AOM Ammonia-oxidizing microorganisms ATP Adenosine triphosphate BOD5 biochemical oxygen demand after five days C- C-terminal Ca. Candidatus COD chemical oxygen demand CV column volume DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid DO dissolved oxygen DOC dissolved organic carbon EDTA Ethylenediaminetetraacetic acid EOS energy optimization of sludge treatment FF Fast flow GOI gene of interest GST Glutathione S-transferase h Hour HAO Hydroxylamine oxidase HURM Hydroxylamine ubiquinone redox module IPTG Isopropyl-β-D-1-thiogalactopyranoside kDa Kilodalton LB Lysogeny broth MBP Maltose-binding protein MCO Multicopperoxidase min Minute N- N-terminal NOB Nitrite-oxidizing bacteria Nxr Nitrite-oxidoreductase ORP oxidation-reduction potential PBS Phosphate-buffered saline rcf Relative centrifugal force rpm Rounds per minute rTCA reductive tricarboxylic acid RT Room temperature s Second SBR sequencing batch reactor SRT sludge/solid retention time T Temperature Tat Twin arginine translocation TN Total nitrogen 50
TOC total organic carbon Trx Thioredoxin VMWWTP Vienna Main Wastewater treatment plant WWTP Wastewater treatment plant YT Yeast – tryptone broth 6H Polyhistidin-tag
7 List of figures Fig. 1: Nitrogen cycle. Figure modified after: (4) Fig. 2: Cytoplasmic and periplasmic oriented Nxr. Figure modified after: (14) Fig. 3: Ammonia oxidation pathways of ammonia-oxidizing archea. Figure modified after: (24) Fig. 4: Crystal structure of cyanase from E. coli. Figure modified after: (41) Fig. 5: SDS-PAGE of chap_pETM33 and chap_pETM44 Fig. 6: SDS-PAGE of cell lysate of MCO_p3NH Fig. 7: SDS-PAGE of cynS_pETM22 and cynS_p3NH Fig. 8: SDS-PAGE of NxrB large-scale expression and purification Fig. 9: Rev-HIS-Trap chromatogram and SDS-PAGE of first purification steps Fig. 10: SEC-elution profile and SDS-PAGE of cynS Fig. 11: SEC-MALS elution profile of cynS Fig. 12: Time series measurement of cynS Fig. 13: Double reciprocal plot of cynS activity
Fig. 14: Representation of inhibitory effect of NaN3 on cyanase
Fig. 15: Double reciprocal plot of cynS inhibited by NaN3 Fig. 16: Nitrogen compounds in wastewater. Figure modified after: (4) Fig. 17: Flow scheme of the Hybrid®-mode. Figure modified after: (69) Fig. 18: Flow scheme of Hybrid® and Bypass-mode. Figure modified after: (59) Fig. 19: Flow scheme WWTP with dewatering and digester units. Figure modified after: (59) Fig. 20: Reaction mechanism of anammox and hydroxylamine to hydrazine. Figure modified after: (81) Fig. 21: Schematic illustration of deammonification. Figure modified after: (75) Fig. 22: Schematic illustration of two-step ANAMMOX® process. Figure modified after: (75) Fig. 23: Schematic illustration of single-step ANAMMOX® process. Figure modified after: (75) Fig. 24: Schematic illustration of DEMON® sequencing batch reactor (Figure modified after: https://www.rmwea.org/docs/Deammonification_PWO_Seminar_20190207.pdf) Fig. 25: Schematic illustration of two zones of a biolayer on a carrier. Figure modified after: (75) Fig. 26: Schematic illustration of a moving bed biofilm reactor. Figure modified after: (92)
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8 List of tables Tab. 1: Cloned Nxr-constructs Tab. 2: Cloned Nxr chaperone-constructs Tab. 3: Cloned MCO-constructs Tab. 4: Cloned cynS-constructs Tab. 5: PCR program Tab. 6: Small-scale expressed Nxr-constructs Tab. 7: Small-scale expressed Nxr chaperone-constructs Tab. 8: Small-scale expressed MCO-constructs Tab. 9: Small-scale expressed cynS-constructs Tab. 10: HisTrap method Tab. 11: Small-scale expressed Nxr-constructs Tab. 12: Small-scale co-expressed Nxr- and Nxr chaperpone- constructs Tab. 13: Small-scale expressed Nxr chaperone-constructs Tab. 14: Small-scale expressed MCO-constructs Tab. 15: Small-scale expressed cynS-constructs Tab. 16: Comparison of different cyanase km-values Tab. 17: Planned Nxr-constructs Tab. 18: Planned MCO-constructs Tab. 19: Overview of WWTP parameters
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